Apparatus and method for fabricating, sorting, and integrating materials with holographic optical traps

ABSTRACT

A method of manufacturing a semiconductor device able to reduce the number of manufacturing steps and attain the rationalization of a manufacturing line is disclosed. The semiconductor device is a high-frequency module assembled by mounting chip parts ( 22 ) and semiconductor pellets ( 21 ) onto each of wiring substrates ( 2 ) formed on a matrix substrate ( 27 ) after inspection. A defect mark ( 2   e ) is affixed to a wiring substrate ( 2 ) as a block judged to be defective in the inspection of the matrix substrate ( 27 ), then in a series of subsequent assembling steps the defect mark (e) is recognized and the assembling work for the wiring substrate ( 2 ) with the defect mark ( 2   e ) thereon is omitted to attain the rationalization of a manufacturing line.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention is a divisional application of U.S. patentapplication Ser. No. 11/029,396, filed Jan. 6, 2005 now U.S. Pat. No.7,588,940, which is a divisional of U.S. patent application Ser. No.10/210,519, filed Aug. 1, 2002, now U.S. Pat. No. 6,863,406, issued Mar.8, 2005, the disclosures of which are incorporated herein by referencein their entirety.

The portion of this invention relating to spatially resolvedphotochemistry using holographic optical traps was made with U.S.Government support provided by the National Science Foundation throughGrant Number DMR-9730189 and by the MRSEC program of the NSF throughGrant Number DMR-9880595. The portion of this invention relating tosorting nonabsorbing from absorbing particles using optical traps wasmade with U.S. Government support provided by the National ScienceFoundation through Grant Number DMR-9730189.

The present invention is related generally to a method and apparatus formanipulating and modifying small dielectric particles or other materialsusing the intense illumination and intensity gradients in stronglyfocused beams of light. In particular, the invention is related to amethod and apparatus which uses focused laser light directed by adiffractive optical element, such as a hologram or diffraction grating,to create optical traps or traps and any one of a variety of selectableoptical intensity patterns to assemble or direct particulate materials,or other affected materials, into a desired spatial pattern for any oneof a myriad of uses. More particularly, the invention is related tomethods for manipulating, effecting interaction of, photochemicallytransforming and/or sorting small dielectric particles or othermaterials.

It is known to construct an optical trap (i.e., trap) using opticalgradient forces from a single beam of light to manipulate the positionof a small dielectric particle immersed in a fluid medium whoserefractive index is smaller than that of the particle. The optical traptechnique has been generalized to enable manipulation of reflecting,absorbing and low dielectric constant particles as well. Likewise, U.S.Pat. No. 6,055,106, co-invented by the inventor named herein andincorporated herein by reference, discloses the manipulation of multipleparticles with multiple traps. However, it was previously unknown to useoptical traps for the various applications of this invention.

Optical traps, originally described by A. Ashkin et al., have become anestablished method for trapping, moving and otherwise manipulatingmesoscopic volumes of matter. See A. Ashkin et al., “Observation ofsingle-beam gradient force optical trap for dielectric particles,”Optics Letters 11, 288-290 (1986). Central to their operation isminimizing the absorption of trapping light to avoid damaging thetrapped material. Optical scalpels operate on the opposite principle,using the energy in a tightly focused laser beam to cut through softmaterials. This application discloses and claims a novel hybrid systemin which focused beams of laser light operate as optical traps for somenonabsorbing particles in a heterogeneous sample and simultaneously asoptical scalpels for others.

Another application of optical trap technology of the invention involvesintroducing foreign materials into living cells by breaching the cellmembrane without causing it to fail entirely, and for moving thematerials through the breach. Various methods for accomplishing thishave been developed, including viral vectors for transfecting shortlengths of DNA, the gene gun and its variants for transferring largersections, and electroporation for inducing transmembrane diffusion. Noneappears to be appropriate for transferring physically large materials,particular if those materials are themselves fragile. The presentmethods and apparatus described herein solves this and other problems.

In addition, holographic optical traps can be used to effectspatially-resolved photochemistry having several advantages overcompeting techniques for chemically defining small structures. Forexample, spatially-resolved photochemistry implemented with opticaltraps facilitates the creation of three-dimensional structures withfeatures ranging in size from a small fraction of the wavelength oflight to macroscopic scales. While techniques such as dip-pennanolithography and microcontact printing offer superior spatialresolution, they are not amenable to three-dimensional fabrication. Avery wide variety of photochemical reactions are known, and any of thesemight be amenable to spatially-resolved photo-fabrication. Thusspatially-resolved photochemistry offers more flexibility than mostmicro- and nano-fabrication methodologies. Performing spatially-resolvedphotochemistry with holographic optical traps greatly enhances theutility of the basic approach by greatly improving its efficiency.

It is therefore an object of the invention to provide an improved methodand system for simultaneously establishing a plurality of optical trapsusing a single and/or plurality of devices, such as, for example,multiple holographic optical trap implementations operatingsimultaneously on a single sample and multiple optical traps andmultiple intensity regions operating simultaneously on a single sample.

It is an additional object of the invention to provide a novel methodand apparatus for using holograms for generating an optical gradientfield for controlling a plurality of particles or other optical media.

It is a further object of the invention to provide an improved methodand system for establishing a plurality of optical traps for a varietyof commercial applications relating to manipulation of small particlessuch as in photonic circuit manufacturing, nanocomposite materialapplications, fabrication of electronic components, opto-electronicdevices, chemical and biological sensor arrays, assembly of holographicdata storage matrices, facilitation of combinatorial chemistryapplications, promotion of colloidal self-assembly, and the manipulationof biological materials.

It is a further object of the invention to provide an improved methodand system for using optical traps to incorporate foreign matter intoliving cells.

It is yet another object of the invention to provided an improved methodand system to sort optically nonabsorbing particles from opticallyabsorbing particles.

It is yet another object of the invention to provide an improved methodand system to implement the fabrication of heterogeneous structuresusing spatially resolved photochemistry.

It is still another object of the invention to provide an improvedmethod and system for constructing a temporally and spatially varyingconfiguration of optical gradient fields for various particle sortingapplications.

It is also an object of the invention to provide a novel method andsystem for using one or more laser beams in conjunction with one or morediffractive optical elements for constructing a selectable time varyingand/or particular spatial array of optical traps for manipulating adielectric metallic materials and other materials.

It is yet a further object of the invention to provide an improvedmethod and system using a single input laser beam, a diffractive opticalelement, and a converging lens to form a static or dynamic optical trapwhich, in conjunction with other so formed optical traps can be used tomanipulate, effect interaction of, photochemically transform and/or sortsmall dielectric particles or other materials.

It is also a further object of the invention to provide an improvedmethod and system employing a laser beam input to a diffractive opticalelement with a beam scanning system enabling scanning of an array ofoptical traps for various commercial applications.

It is in addition another object of the invention to provide a novelmethod and apparatus for constructing an optical trap configurationusing a laser beam, a diffractive optical element and a convergingoptical system to form the trap configuration at a selectable locationrelative to an objective lens focal plane.

It is yet another object of the invention to provide a novel method andapparatus for using a laser beam input to a diffractive optical elementto generate a three-dimensional arrangement of optical traps.

It is another object of the invention to provide a novel method forcreating multiple independently steered optical traps using atime-dependent addressable phase-shifting medium (such as a liquidcrystal phase shifting array or other phase medium) as a diffractiveoptical element.

It is a further object of the invention to provide a novel method forcreating time-dependent optical gradient fields for the segregation ofmicroscopic particles.

It is yet another object of the invention to provide a novel method formanipulating a plurality of biological objects including thecrystallization of proteins or implementing other phase changes.

Other objects, features and advantages of the present invention will bereadily apparent from the following description of the preferredembodiments thereof, taken in conjunction with the accompanying drawingsdescribed below wherein like elements have like numerals throughout.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a prior art method and system for a single opticaltrap;

FIG. 2 illustrates a prior art method and system for a single, steerableoptical trap;

FIG. 3 illustrates a method and system using a diffractive opticalelement;

FIG. 4 illustrates another method and system using a tilted opticalelement relative to an input light beam;

FIG. 5 illustrates a continuously translatable optical trap (trap) arrayusing a diffractive optical element;

FIG. 6 illustrates a method and system for manipulating particles usingan optical trap array while also forming an image for viewing theoptical trap array;

FIG. 7A illustrates an image of a four by four array of optical (traps)using the optical system of FIG. 6; and FIG. 7B illustrates an image ofone micrometer diameter silica spheres suspended in water by the opticaltraps of FIG. 7A immediately after the trapping illumination has beenextinguished, but before the spheres have diffused away;

FIG. 8A illustrates a first step in transferring material into a cellwith material encapsulated in a liposome being immobilized with opticaltraps; FIG. 8B illustrates a liposome being fused to a cell membrane;and FIG. 8C the material in the liposome is transferred through a breachin the liposome cell function; and

FIG. 9 is a functional block flow diagram illustrating separation ofnonabsorbing from absorbing particles.

DETAILED DESCRIPTION OF THE INVENTION

This invention presents several uses for the “Apparatus for ApplyingOptical Gradient Forces” disclosed and claimed in U.S. Pat. No.6,055,106 to Grier et al. That apparatus is encompassed by use of theterminology optical trap, optical trap and optical gradient force traphereinafter. By way of introduction, FIGS. 1 and 2 illustrate severalprior art methods and systems. These systems will first be reviewed, andthen the methods of the present invention will be described in terms ofthe optical trap embodiment examples of FIGS. 3-7A and 7B. In prior artoptical trap system 10 of FIG. 1, optical gradient forces arise from useof a single beam of light 12 to controllably manipulate a smalldielectric particle 14 dispersed in a medium 16 whose index ofrefraction is smaller than that of the particle 14. The nature of theoptical gradient forces is well known, and also it is well understoodthat the principle has been generalized to allow manipulation ofreflecting, absorbing and low dielectric constant particles as well.

The optical trap system 10 is applied by using a light beam 12 (such asa laser beam) capable of applying the necessary forces needed to carryout the optical trapping effect needed to manipulate a particle. Themethod used to create a conventional form of the optical trap 10 is toproject one or more beams of light, each with a specified degree ofcollimation, through the center of a back aperture 24 of a convergingoptical element (such as an objective lens 20). As noted in FIG. 1 thelight beam 12 has a width “w” and having an input angle, .PHI., relativeto an optical axis 22. The light beam 12 is input to a back aperture 24of the objective lens 20 and output from a front aperture 26substantially converging to a focal point 28 in focal plane 30 ofimaging volume 32 with the focal point 28 coinciding with an opticaltrap 33. In general, any beam of light brought to a diffraction-limitedfocus, and possessing sufficiently large axial intensity gradients totrap a particle stably against axial radiation pressure, can form thebasis for the optical trap system 10.

Creating such a focus requires a focusing element with sufficiently highnumerical aperture and sufficiently well-corrected aberrations.Generally, the minimum numerical aperture to form a trap is about 0.9 toabout 1.0.

In the case of the light beam 12 being a collimated laser beam andhaving its axis coincident with the optical axis 22, the light beam 12enters the back aperture 24 of the objective lens 20 and is brought to afocus in the imaging volume 32 at the center point c of the objectivelens focal plane 30. When the axis of the light beam 12 is displaced bythe angle .PHI. with respect to the optical axis 22, beam axis 31 andthe optical axis 22 coincide at the center point B of the back aperture12. This displacement enables translation of the optical trap across thefield of view by an amount that depends on the angular magnification ofthe objective lens 20. The two variables, angular displacement .PHI. andvarying convergence of the light beam 12, can be used to form theoptical trap at selected positions within the imaging volume 32. Amultiple number of the optical traps 33 can be arranged in differentlocations provided that multiple beams of light 12 are applied to theback aperture 24 at the different angles .PHI. and with differingdegrees of collimation.

In order to carry out optical trapping in three dimensions, opticalgradient forces exerted on the particle to be trapped must exceed otherradiation pressures arising from light scattering and absorption. Ingeneral this necessitates the wave front of the light beam 12 to have anappropriate shape at the back aperture 24. For example, for a GaussianTEM₀₀ input laser beam, the beam diameter, w, should substantiallycoincide with the diameter of the entrance pupil 24. For more generalbeam profiles (such as Laguerre-Gaussian modes) comparable conditionscan be formulated.

In another prior art system in FIG. 2, the optical trap system 10 cantranslate the optical trap 33 across the field of view of the objectivelens 20. A telescope 34 is constructed of lenses L1 and L2 whichestablishes a point A which is optically conjugate to the center point Bin the prior art system of FIG. 1. In the system of FIG. 2 the lightbeam 12 passing through the point A also passes through the point B andthus meets the basic requirements for performing as the optical trapsystem 10. The degree of collimation is preserved by positioning thelenses L1 and L2 as shown in FIG. 2, their focal lengths and otheroptical characteristics being selected to optimize the transferproperties of the telescope 34. In particular, the magnification of thetelescope 34 can be chosen to optimize angular displacement of the lightbeam 12 and its width w in the plane of the back aperture 24 of theobjective lens 20. As stated hereinbefore, in general several of thelight beams 12 can be used to form several associated optical traps.Such multiple beams 12 can be created from multiple independent inputbeams or from a single beam manipulated by conventional reflectiveand/or refractive optical elements.

In one optical trap configuration, shown in FIG. 3, arbitrary arrays ofoptical traps can be formed. A diffractive optical element 40 isdisposed substantially in a plane 42 conjugate to back aperture 24 ofthe objective lens 20. Note that only a single diffracted output beam 44is shown for clarity, but it should be understood that a plurality ofsuch beams 44 can be created by the diffractive optical element 40. Theinput light beam 12 incident on the diffractive optical element 40 issplit into a pattern of the output beam 44 characteristic of the natureof the diffractive optical element 40, each of which emanates from thepoint A. Thus the output beams 44 also pass through the point B as aconsequence of the downstream optical elements described hereinbefore.In some situations, where it is desired to create a plurality of objectsin a specific spatial relationship to one another, with each object in aspecific orientation, it will be necessary to create the plurality ofobjects on a timescale faster than that on which relevant motion of theobjects occurs. This timescale will be a function of, among otherfactors, the viscosity of the medium. In such a situation, an apparatuswhich allows fabrication of the plurality of objects in parallel mayprovide an advantage over one which fabricates the objects sequentially.

The diffractive optical element 40 of FIG. 3 is shown as being normal tothe input light beam 12, but many other arrangements are possible. Forexample, in FIG. 4 the light beam 12 arrives at an oblique angle βrelative to the optic axis 22 and not at a normal to the diffractiveoptical element 40. In this embodiment, the diffracted beams 44emanating from point A will form optical traps 50 in focal plane 52 ofthe imaging volume 32 (seen best in FIG. 1). In this arrangement of theoptical trap system 10 an undiffracted portion 54 of the input lightbeam 12 can be removed from the optical trap system 10. Thisconfiguration thus enables processing less background light and improvesefficiency and effectiveness of forming optical traps.

The diffractive optical element 40 can include computer generatedholograms which split the input light beam 12 into a preselected desiredpattern. Combining such holograms with the remainder of the opticalelements in FIGS. 3 and 4 enables creation of arbitrary arrays in whichthe diffractive optical element 40 is used to shape the wavefront ofeach diffracted beam independently. Therefore, the optical traps 50 canbe disposed not only in the focal plane 52 of the objective lens 20, butalso out of the focal plane 52 to form a three-dimensional arrangementof the optical traps 50.

In the optical trap system 10 of FIGS. 3 and 4, also included is afocusing optical element, such as the objective lens 20 (or other likefunctionally equivalent optical device, such as a Fresnel lens) toconverge the diffracted beam 44 to form the optical traps 50. Further,the telescope 34, or other equivalent transfer optics, creates a point Aconjugate to the center point B of the previous back aperture 24. Thediffractive optical element 40 is placed in a plane containing point A.

In another embodiment, arbitrary arrays of the optical traps 50 can becreated without use of the telescope 34. In such an embodiment thediffractive optical element 40 can be placed directly in the planecontaining point B. In another form of the invention, one of the lensescan be positioned in the hologram itself rather than in the telescope34.

In the optical trap system 10 either static or time dependentdiffractive optical elements 40 can be used. For a dynamic, or timedependent version, one can create time changing arrays of the opticaltraps 50 which can be part of a system utilizing such a feature. Inaddition, these dynamic optical elements 40 can be used to actively moveparticles and other materials with diverse optical properties relativeto one another. For example, the diffractive optical element 40 can be aliquid crystal spatial light modulator encoding computer-generated phasemodulations onto the wavefront of an incident laser beam. In anotherembodiment, a spatial light modulator may also be used in conjunctionwith a phase ring in place of the diffractive optical element.

In another embodiment illustrated in FIG. 5, a system can be constructedto carry out continuous translation of the optical trap 50. A gimbalmounted mirror 60 is placed with its center of rotation at point A. Thelight beam 12 is incident on the surface of the mirror 60 and has itsaxis passing through point A and will be projected to the back aperture24. Tilting of the mirror 60 causes a change of the angle of incidenceof the light beam 12 relative to the mirror 60, and this feature can beused to translate the resulting optical trap 50. A second telescope 62is formed from lenses L3 and L4 which creates a point A′ which isconjugate to point A. The diffractive optical element 40 placed at pointA′ now creates a pattern of diffracted beams 64, each of which passesthrough point A to form one of the trap 50 in an array of the opticaltraps system 10.

In operation of the embodiment of FIG. 5, the mirror 60 translates theentire trap array as a unit. This methodology is useful for preciselyaligning the optical trap array with a stationary substrate, fordynamically stiffening the optical trap 50 through small-amplitude rapidoscillatory displacements, as well as for any application requiring ageneral translation capability.

The array of the optical traps 50 also can be translated verticallyrelative to the sample stage (not shown) by moving the sample stage orby adjusting the telescope 34. In addition, the optical trap array canalso be translated laterally relative to the sample by moving the samplestage. This feature would be particularly useful for movement beyond therange of the objective lens' field of view.

In another embodiment shown in FIG. 6 the optical system is arranged topermit viewing images of particles trapped by the optical traps 10. Adichroic beamsplitter 70, or other equivalent optical beamsplitter, isinserted between the objective lens 20 and the optical train of theoptical trap system 10. In the illustrated embodiment the beamsplitter70 selectively reflects the wavelength of light used to form the opticaltrap array and transmits other wavelengths. Thus, the light beam 12 usedto form the optical traps 50 is transmitted to the back aperture 24 withhigh efficiency while light beam 66 used to form images can pass throughto imaging optics (not shown).

In yet another embodiment of the invention a method for incorporatingforeign matter into living cells is described. It has been determinedrecently that optical trap devices can be advantageously used toincorporate foreign matter such as an artificial chromosome, into livingcells using a combination of optical trapping, optically inducedmembrane fusion and optical cutting. By way of nonlimiting example, themethod includes the steps of encapsulating the material to betransferred in, for example, a liposome, fusing the liposome to the cellmembrane, and puncturing the juncture to effect transfer. The first steptakes advantage of any of a variety of known possible encapsulationtechniques. Once encapsulation is complete, the liposome can be capturedwith optical traps and translated toward a target cell. Depending on thematerial's sensitivity to light, several separate optical traps might bepreferable to one, in which case holographic optical traps offeradvantages to other techniques, such as scanned optical traps.

Unlike scanned optical traps which address multiple trapping points insequence, and thus are time-shared, holographic optical traps illuminateeach of their traps continuously. For a scanned optical trap to achievethe same trapping force as a continuously illuminated trap, it mustprovide at least the same time-averaged intensity. This means that thescanned trap has to have a higher peak intensity by a factorproportional to at least the number of trapping regions. This higherpeak intensity increases the opportunities for optically-induced damagein the trapped material. This damage can arise from at least threemechanisms: (1) single-photon absorption leading to local heating, (2)single-photon absorption leading to photochemical transformations, and(3) multiple-photon absorption leading to photochemical transformations.Events (1) and (2) can be mitigated by choosing a wavelength of lightwhich is weakly absorbed by the trapping material and by the surroundingfluid medium. Event (3) is a more general problem and is mitigated inpart by working with longer-wavelength light. Multiple-photonabsorption, the central mechanism of the photopolymerization part ofthis disclosure, occurs at a rate proportional to the intensity raisedto a power (i.e., I² for two-photon absorption). The rates for suchprocesses are rapidly reduced to acceptable levels by reducing the peakintensity of the trapping beam. As a result, lower intensity,continuously-illuminated holographic optical traps are preferable totime-shared scanned traps. Furthermore, the holographic optical trapmethod lends itself to distributing more independent traps throughoutthe volume of an extended object than does any scanned trap technique.In particular, holographic optical traps can distribute traps across anobject's three-dimensional contours, unlike scanned traps which arelimited to a single plane.

Distributing the trapping force among multiple sites on an objectfurther permits holographic optical traps to minimize the maximumintensity and maximum force applied to any one point of the object. Thismay be thought of as being analogous to a bed of nails, in which any onenail could cause damage, but distributing the loading among multiplenails reduces the local force below the threshold for damage.

Consequently, holographic optical traps offer substantial benefits overboth scanned traps and individual conventional optical traps. If thecell itself is motile, it also may be held in place and oriented withholographic optical traps. For some applications, for example whenmaterial must be transferred to a particular part of a cell whilebypassing others, optical trap manipulation offers advantages. A singleset of holographic optical traps can be used to hold both the cell andthe liposome simultaneously.

As shown in FIG. 8C a cell 200 has an impermeable wall 210, as forexample in a plant cell. An optical scalpel can be used to cut awayenough of the wall 210 to expose a region of cell membrane 215 forsubsequent liposome fusion. The laser used for this cutting or ablationmost likely will operate at a shorter wavelength than that used forholding and moving a liposome 220 and the cell 200. Unlike trapping,where material damage is usually undesirable, cutting requires stronginteraction between the focused light and the material. Consequently,the conditions discussed above for minimizing damage also provide aguide to optimizing desired damage. In particular, shorter wavelengthlight carries more energy per photon than longer wavelength light. Eachphoton absorption therefore is more likely to deliver enough energy todisrupt chemical bonds and to rearrange macromolecules in the cell wall210 and the cell membrane 215. The rate of all such transformations isincreased in shorter wavelength light.

Once an appropriate section of the cell membrane 215 has been exposed,the liposome 220 can be moved into proximity, again using optical trapsforces (see FIG. 8A). Fusion can be accomplished either chemically,through the action of proteins or other biochemical agents incorporatedinto the liposome's outer leaf, or optically through one or more pulsesof light directed at the liposome-membrane interface (see FIG. 8B).

Fusion can proceed to effect the transfer in one step, or else furtherchemical treatment or additional pulse of light may be required tobreach the membrane-liposome interface. Once the interface is breached,the liposome's contents (material 240) can transfer into the cell 200through diffusion, or else can be moved into the cell 200 with one ormore of the optical traps. In addition, for artificial chromosomes, forexample, the material 240 can be placed directly into cell nucleus 220by using the optical traps to transfer the matter through the cellmembrane 215 and cytoplasm and, thereafter, cutting the nuclear membraneto effect transfer into the nucleus 220 directly.

Once transfer is complete, the cell 200 can be held in place for furtherobservation before being collected. Both holding and collection can befacilitated by optical trap manipulation, particularly if the entireprocess described above takes place in a closed microfluidic system.

The entire process, from sample selection to cell collection can becarried out using a conventional light microscope for observation.Indeed, the same optical train used to create the optical traps andscalpel for this process also can be used to monitor its progress. If,furthermore, all steps are carried out using holographic optical traps,or a related manipulation technique, then the entire process also can beautomated, with digitally recorded microscope images being used toprogram the pattern of optical traps and their motions.

The substance or the material 240 to be introduced into the cell 200 canbe any substance and will preferably not be endogenous to the cell 200into which it is to be introduced. Preferably the substance is asubstance not normally able to cross the cell membrane. It is preferredthat the substance to be introduced into the cell 200 is a hydrophilicsubstance, however the substance may also be hydrophobic. Any biologicalmolecule or any macromolecule, for example, a complex of molecules, canbe introduced into the cell 200. The material 240 generally has amolecular weight of 100 daltons or more. In a more preferred embodiment,the material 240 is a nucleic acid molecule such as DNA, RNA, PNA (e.g.cDNA, genomic DNA, a plasmid, a chromosome, an oligonucleotide, anucleotide sequence, or a ribozyme) or a chimeric molecule or a fragmentthereof, or an expression vector. Additionally, the material 240 may beany bio-active molecule such as a protein, a polypeptide, a peptide, anamino acid, a hormone, a polysaccharide, a dye, or a pharmaceuticalagent such as drug.

Although this discussion has focused on methods for modifying a singlecell using the contents of a single liposome, the same approach could beused to fuse multiple liposomes to a single cell, and to processmultiple cells simultaneously.

In another form of the invention a system and method are provided forsorting nonabsorbing particles from absorbing particles 290 isconstructed (see FIG. 9). It has been discovered that an optical trap ortrap array 300 can be advantageously formed from focused beams of laserlight which operate as optical traps for some nonabsorbing particles 310in a sample and as optical scalpels for others. Rather than preciselycutting the absorbing particles 290 as traditionally done by an opticalscalpel, however, absorption of light is used to obliterate theabsorbing particles 290 nonspecifically so as to reduce them to verysmall pieces 330. These small pieces then can be separated from theundamaged nonabsorbing particles left behind in optical traps 320.

An example of the utility of this method is the problem of searching forcancerous cells in a sample of blood. Ordinarily, the vast number of redblood cells in the sample would have to be separated from the candidatecancer cells before testing can begin. Light from optical trapsoperating in the visible range of wavelengths, for example at awavelength of 532 nm, would be absorbed strongly by red blood cells andconsequently can be used to destroy them through local heating. Otherunpigmented cells, however, can be trapped by the same visible traps andmanipulated for further testing. Consider, for example, an array ofvisible optical traps arranged with their characteristic spacingconsiderably smaller than the size of a red blood cell. A mixture ofcells driven through this array of optical traps by an externallymediated fluid flow would encounter these optical traps. Thestrongly-absorbing cells would be reduced to much smaller components,such as membrane fragments through their interaction with the light.These smaller components would have a comparatively weaker interactionwith the light and a small portion might be trapped by some of the trapsin the array. More likely, however, they would be washed away by thefluid flow. Rather than being damaged by the light, weakly absorbingcells would encounter one or more optical traps in the array andexperience a trapping force.

The intact cells would have larger and more numerous regions susceptibleto optical trapping than the fragments of the destroyed cells, andtherefore would be preferentially trapped by the array of optical traps.Cells localized in the array of optical traps can be transported forcollection by moving the optical traps themselves, for example takingadvantage of the features of an earlier application of the assigneeherein, (Grier et al., application Ser. No. 09/875,812 which isincorporated by reference herein.) by moving the sample container totransport the trapped cells to a collection region within the samplecontainer, or by periodically turning off the traps and directing thecells through a flow of fluid to a collection area. In any of theseways, the cells which do not absorb light are collected separately fromthe cells that do.

This approach can be generalized from sorting cells to sorting any othermaterial whose absorption coefficients differ substantially for at leastone particular wavelength of light. The benefits of this manipulationinclude excellent fidelity for rejecting the undesired absorbingmaterial, and the ability to perform other active sorting steps. Thesame benefits would accrue to other applications of this ablativeparticle sorting method.

In preferred embodiments of optically ablative particle sorting,separation of nonabsorbing particles can be effected with multipleoptical traps created with the holographic optical trap technique.Separation of the trapped particles from the obliterated absorbingparticles could be performed with the previously disclosed techniques ofactive trap manipulation, optical peristalsis, or passive lateraldeflection in a flow. The separation could also be performed in amicrofluidics device with one channel for flushing waste products fromthe obliteration of absorbing particles and other channels forcollecting selected nonabsorbing particles.

In previous uses of optical traps, great care was required to select awavelength of light which would not damage any of the material to betrapped. In the present invention the goal is to select a wavelengthwhich is absorbed strongly by the unwanted subpopulation of a mixedsample, and very weakly by the other subpopulation to be retrieved.Retrieval of the weakly absorbing subpopulation proceeds throughconventional methods, and the separation in this case being effectedthrough the passive destruction of the unwanted fraction, rather thanthrough active selection. This could also be a preprocessing step forother analytical methods such as flow cytometry.

By way of nonlimiting example, this method could be used for earlydetection of cancer through blood screening. To with, several kinds ofcancers in their earliest stages do not form particularly well definedtumors but, instead, define regions of abnormal cells which tend toslough into the bloodstream. In practice, detection of those cells wouldprovide an indication that the patient has an early stage cancer. Suchdetection would provide at least a tentative diagnosis long before othermethods requiring detection of a complete tumor or its metabolicproducts. Thus, this method would provide for early and more effectivetreatment. This can be compared with conventional separation methods ofcentrifugation to separate the denser, hemoglobin-bearing red bloodcells from other cells carried in the blood. However, centrifugationoften entrains the lighter cells with the heavier ones, thus makingdetection very difficult.

Using the method of the present invention, blood samples can be made toflow through an array of optical traps having wavelength and intensitythat will destroy the cellular structure of the red blood cells, leavingnon-red cells, such as white blood cells and possible cancer cells,intact. In fact, the red blood cells will be reduced to fragments toosmall to trap. In contrast, the undamaged cells can be trapped by theoptical traps and transported, for example, by sequentially updating thepattern of traps, to a collection point for subsequent analysis.

In yet another embodiment of the invention a method concernsimplementing spatially resolved photochemistry. Light can provide theactivation energy for photochemical reactions, and in cases where onephoton does not carry enough energy to initiate a photochemicalreaction. The photochemical reaction still can proceed if two or morephotons are absorbed simultaneously, such that the combined energy ofall absorbed photons exceeds the activation threshold for the reaction.The rate at which multi-photon processes proceed depends nonlinearly onthe intensity of the available light, with two-photon absorptionoccurring at a rate proportional to l², the square of the light'sintensity. This nonlinear dependence on intensity can be used toinitiate photochemical reactions only in selected volumes within alarger sample and to proceed in a spatially resolved manner. Thereaction only takes place in regions which are illuminated sufficientlyintensely, and not in others.

Optical traps are tightly focused beams of light and, therefore, offeran ideal method for producing spatially resolved structures throughphotochemistry. The focal point in an optical trap is the most intenseregion of the illumination field. Tuning the intensity of this focalregion close to the threshold for an appreciable rate of photochemicaltransformation facilitates controlled photochemistry in a volumecomparable to the diffraction-limited focal volume of the optical trap.Whereas optical traps generally are used to trap and manipulate smallvolumes of matter, here they are being used to transform matter indesirable ways. Single optical traps have been used in the art to createlocally intense illumination for initiating and propagating two-photonphotochemistry to create photopolymerized devices as small as 10micrometers in diameter. Defining photochemical patterns in previousconventional methods required either translating a single optical trapthrough the fluid precursor, or translating the fluid past the singlestationary trap. In either case, the process of creating a structure byspatially-resolved photochemistry involved sequentially illuminatingtarget volumes.

Unlike prior methods the present invention uses multiple holographicoptical traps to perform spatially resolved photochemistry at multiplelocations simultaneously to create structures composed of eitherheterogeneous or homogeneous materials. For example, in previousmethods, one can use the multiple beams to draw multiple copies of thesame structure at once, thus allowing the fabrication of multipleidentical structures simultaneously. Alternatively, one could usemultiple beams of light to simultaneously create different aspects of asingle structure, thus allowing it to be made much more rapidly.Finally, separate beams can be used to create the outside structurearound an extended volume and to simultaneously created interior volumestructures (i.e., structures inside the separately created shell aroundthe volume). Common to all of those techniques is the creation of astructure constituted from a homogeneous material such as a gel. Here,unlike the prior art, the unique combination of the manipulation of theoptical traps and the chemical transformations effected thereby, alsopermit the creation of single or multiple heterogeneous structures. Forexample, where certain objects are preformed, particular optical trapscan be used to hold them in place while other similarly focused beams oflight are used to create interconnections made of photochemicallytransformed materials, thereby creating heterogeneous structures.

Moreover, unlike conventional optical traps, holographic optical trapsuse computer-generated diffractive optical elements to define multipleoptical traps in any user-specified pattern in three dimensions. Eachfocal point in such a trapping pattern can be used to inducephotochemical transformations. Computer algorithms permit placement ofone or more optical traps anywhere within a three-dimensional accessiblevolume, and also permit independent modification of the properties ofeach of those traps. Creating a new configuration with the traps at alocation displaced from the old trap, or where the trap properties areslightly different, can be effected by calculating and projecting a newhologram. A fixed arrangement of traps therefore can be steered througha precursor solution to fabricate multiple copies of aphotochemically-defined pattern, although a sequence of small stepsmight be required to effect large changes. Conversely, the individualtraps in a holographic optical trap array can be moved independently bycalculating and projecting a sequence of computer-generated diffractionpatterns with each trap's position updated as required in each pattern.This would enable multiple traps to induce photochemical transformationsin multiple regions simultaneously and would be useful for efficientlyaddressing multiple parts of one or more photochemically-definedstructures. Benefits of the holographic optical trap technique in theseapplications include greatly improved throughput, and the opportunity totailor initiation and growth propagation rates locally so as to optimizematerial properties in the finished product which might depend on suchaspects of the formation process.

In the method of the invention the holographic optical traps areutilized by photopolymerizing Norland Type 73 UV-cured adhesive andNorland type 88 UV-cured adhesive using light of wavelength 532 nmobtained from a frequency-doubled Nd:YVO₄ laser. We also have usedoptical traps to photopolymerize polyacrylamide from a precursorsolution containing a UV-excited photoinitiator and a free-radicalinhibitor.

While preferred embodiments of the invention have been shown anddescribed, it will be clear to those skilled in the art that variouschanges and modifications can be made without departing from theinvention in its broader aspects as set forth in the claims providedhereinafter.

What is claimed is:
 1. A method for simultaneously producing multiplespatially resolved structures through photochemistry on a material usingat least one of a plurality of holographic optical traps and anillumination spot in conjunction with an optical trap comprising thesteps of; providing a diffractive optical element for receiving a laserbeam and forming a plurality of separate laser beams; providing afocusing element downstream from said diffractive optical element, whichcooperates with said focusing element to separately converge each of thelaser beams to form a separate optical trap for processing at least oneparticle; providing a computer executing a program to control placementof the optical trap within a three-dimensional accessible volume and toprovide independent modification of the optical properties of theoptical trap; and tuning the intensity of the separate laser beams toachieve selected photochemical transformation of the material tofacilitate controlled photochemistry in a volume associated with adiffraction-limited focal volume of the optical trap and an illuminationspot, and manipulating the location and intensity of at least one of theoptical trap and the illumination spot with one of the optical traps toselectively induce photochemical transformations in multiple regions tothereby fabricate multiple copies of a photochemically-defined pattern.2. The method as defined in claim 1 including the step of definingmultiple functions for a plurality of the optical trap in auser-specified pattern in three dimensions.